a review of the key genetic tools to assist imperiled species conservation: analyzing...

A review of the key genetic tools to assist imperiled species conservation: analyzing West Indian manatee populations

Robert K. Bonde1, Peter M. McGuire2, and Margaret E. Hunter1

1Sirenia Project, Southeast Ecological Science Center, U.S. Geological Survey, 7920 NW 71st Street, Gainesville, Florida, 32653, USA 2Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Box 100144,

Gainesville, Florida, 32610, USA

Abstract Managers faced with decisions on threatened and endangered

wildlife populations often are lacking detailed information about the

species of concern. Integration of genetic applications will provide

management teams with a better ability to assess and monitor

recovery efforts on imperiled species. The field of molecular

biology continues to progress rapidly and many tools are currently

available. Presently, little guidance is available to assist researchers

and managers with the appropriate selection of genetic tools to

study the status of wild manatee populations. We discuss several

genetic tools currently employed in the application of conservation

genetics, and address the utility of using these tools to determine

population status to aid in conservation efforts. As an example,

special emphasis is focused on the endangered West Indian manatee

(Order Sirenia). All four extant species of sirenians are imperiled

throughout their range, predominately due to anthropogenic sources;

therefore, the need for genetic information on their population status

is direly needed. [JMATE. 2012;5(1):8-19]

Keywords: Conservation, Management, Genetics, Endangered

Species, Manatee

Introduction

Conservation Genetics and Sirenian Conservation

Conservation genetics (the study of genetic

methods and how they relate to biodiversity in

populations) can inform managers of the optimal actions

needed to conserve these species. Some species have

demonstrated successful adaptations to meet

environmental challenges through random gene

mutations. The natural evolutionary processes of a

wildlife population may also be affected by

anthropogenic impacts to the environment, as well as the

well-intended efforts of wildlife managers (54).

Conservation has a broad definition but for our

discussion it is defined as preservation of a target species

to persist through time. Conservation incorporates a

great deal more than basic science, and the availability

of data does not automate complex conservation actions.

However, society often must make decisions regarding

wildlife populations based upon the best available

science. More rigorous and detailed genetic studies will

assist managers in making policy decisions that could

have long-term consequences for a species. The

conservation and recovery of imperiled wildlife may

require biologically and legally defensible delineation of

management units (MUs), distinct population segments

(DPSs), and subspecies and species designations (28,

79). Additionally, threats analyses must justify that

smaller segments or portions of a population be

designated for fine-scale protective measures.

Genetic data have been instrumental in the

determination of listing status for several endangered

species (19). The data from genetics, coupled with other

demographic sources of information (life history

parameters, abundance, distribution, habitat, etc.), are

necessary for determining which population units or

stocks will benefit from applied management decisions

(18). A recent study on the genetic tools used to assess

threatened and endangered populations of animals aptly

illustrated that when multiple sources of genetic data

were used in concert, there was a higher probability for

the species to receive protection (19).

Some especially helpful techniques employed in

conservation genetics include the use of coding (genes)

or non-coding DNA sequences which are used as

markers to identify individuals, differentiate

populations, provide pedigree information, or examine

functional genomic differences. Within the

mitochondrial genome, a number of regions have been

valuable tools in the field of conservation genetics.

Markers such as the control region and cytochrome-b

have been implemented in phylogeography and

phylogenetic studies. Cytochrome oxidase 1 is used for

‘bar-coding’ or determining species identity with a

single, conserved marker (74). In the nuclear genome,

neutral microsatellite loci can provide contemporary

information on which landscape and environmental

Received May 9, 2012; Accepted July 28, 2012

Correspondence: Robert K. Bonde

Sirenia Project, Southeast Ecological Science Center, U.S. Geological

Survey, 7920 NW 71st Street, Gainesville, Florida, 32653.

Email: [email protected]

Journal of Marine Animals and Their Ecology Copyright © 2008 Oceanographic Environmental Research Society

Vol 5, No 1, 2012 Printed in Canada

JMATE 8

mailto:[email protected]

factors are influencing population structure.

High-throughput sequencing technology can

identify genes for molecular systematics and functional

genomic analysis, as well as enable marker development

(52). Examination of functional markers can enhance

genetic research by providing information to compare

the distinctiveness of individual populations, identify

specific genes and their regulation, and examine the

organism’s response to environmental change.

One case-in-point of an imperiled species that

merits increased attention from conservation genetics is

the manatee. Manatees are large, aquatic, generalist

herbivores utilizing primarily tropical habitats (Figure

1). The Order Sirenia is represented by two Families, the

Trichechidae and the Dugongidae (67). The three extant

species in the family Trichechidae have a wide range on

both sides of the Atlantic Ocean (Figure 2). The

Amazonian manatee (Trichechus inunguis) occupies the

Amazon River Basin, while the West African manatee

(T. senegalensis) inhabits nine coastal countries along

the Atlantic coast of Africa. The oft-researched West

Indian manatee (T. manatus) is divided into two

subspecies, the Florida manatee (T. manatus latirostris)

occupying the southeastern United States and the

Antillean manatee (T. manatus manatus), present

throughout the Caribbean and along coastal Central and

South America through Brazil. The only extant

representative of the family Dugongidae is the dugong

(Dugong dugon) which has a more pandemic

distribution, occupying much of the tropical waters of

the Indian Ocean from East Africa to Australia. A sister

taxa in the Dugongidae, the Steller’s sea cow, occupied

the Bering Sea until it was extirpated in the mid 1700’s

by fur sealers searching for foodstuffs during voyages

(72).

Management and conservation of sirenian

populations would benefit from multiple methods of

genetic population analyses. Utilizing multiple genetic

tools has strengthened the position for implementation

of conservation measures in other threatened and

endangered species as well (19). Due to their cryptic

existence and avoidance of people, manatee behavior

outside of Florida is seldom studied. Genetic data could

provide insight into behaviors and stochastic

demography, including mate selection, reproductive

success, and migration patterns. Mitochondrial DNA

(mtDNA) sequencing, multi-locus analyses utilizing

microsatellites and single nucleotide polymorphisms

(SNPs), gene expression analysis, and genomic

sequencing are the major techniques currently employed

in conservation genetics and could benefit sirenian

populations.

Why Study Manatees?

Emigration through migration and dispersal

has played an important role in manatees’ ability to

adapt and respond to survival challenges, and

accounts for their broad geographic distribution.

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Genetic tools for conservation

Figure 1: Florida manatee in Kings Bay, Crystal River, Florida.

(Photo courtesy of USGS, Sirenia Project).

Figure 2: Sirenian global distribution. Red is Steller’s sea cow

(extinct), pink is dugong, light blue is Amazonian manatee, dark

blue is West Indian manatee, and green is West African manatee.

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resist an extreme environmental challenge. Any

reductions in the population size will likely result in loss

of genetic resistance, making them more vulnerable to

future perturbations. Understanding the relationship

between manatee genetics and manatee behavioral

responses and adaptive capabilities will help managers

direct the recovery of the species.

The consensus among researchers is that the

Florida manatee population has grown in recent decades

and this indicates that the manatee population in Florida

appears to be doing well. However, evidence

documenting a low genetic diversity (23, 36, 56, 62, 77,

82) suggests that the manatee population may not be as

fit as predicted based on just the estimated number of

individuals. In other mammalian species a lack of allelic

diversity has resulted in deleterious effects (22). This

was aptly illustrated when the Florida panther population

was pressed to the verge of extinction resulting in drastic

measures to restore genetic fitness (40). Still, some

wildlife populations, such as the northern elephant seal,

have been able to thrive despite severe historical genetic

bottlenecks resulting in low allelic diversity (33, 71).

Whether this will be the case for the Florida manatees

has yet to be determined. However, the recent increase

in census size is promising for the maintenance of the

current genetic diversity, thus providing some adaptive

advantage into the future.

Genetic Markers

A host of genetic markers and tools are available

to researchers to aid in determining population status.

The selection of the appropriate marker depends on

several variables and on the animal under investigation.

Considerations for proper selection criteria include three

primary factors: (1) Is the selected marker (or markers)

appropriate to answer the questions at hand? (2) Are

there limitations related to sample size and method of

preservation? and (3) What are the budget and time

restrictions, if any? Many analyses are expensive, but

with recent technological advances, the cost has been

reduced. The number of genetic analyses available to the

research community is great. Here we provide

information on the tools that have been employed in the

past to better understand manatee populations and offer

suggestions on methods to help enhance those studies.

Several techniques are discussed in more detail below.

10

Dispersal is also necessary for gene flow and can

increase genetic diversity. West Indian manatees have

persisted in environments where more fragile wildlife

populations like the Florida panther, Florida red wolf,

pallid beach mouse, and Caribbean monk seal have not

thrived or have been completely extirpated due to human

interactions (38, 80). The Steller’s sea cow was forced to

extinction by unregulated hunting as early as the middle

1700’s (72), thus providing evidence of human impacts

on a population with a very limited distribution. Because

of legal protections and the increasing appreciation for

wildlife by humans, Florida manatees have adapted

recently to tolerate and coexist with human populations.

Populations of Antillean manatees are much more

secretive due to direct threats related to historical and

continued hunting (59, 66). The geographical separation

of this species has resulted in neutral and likely

functional genetic differences between the subspecies

(35, 56, 82).

It is likely that all manatee populations have

undergone significant fluctuations, due to natural and

anthropogenic events, that have resulted in low allelic

diversity when compared to other wildlife populations

(57, 58). The low genetic diversity observed in previous

studies on manatees (23, 36, 56, 62, 77, 82) might have

placed limits on their ability to cope with some

environmental and anthropogenic changes, but those

limitations appear not to have affected fecundity and

survival of the Florida subspecies. To achieve a better

understanding of the low diversity and how that impacts

fecundity between stocks of manatees, efforts should

focus on immune and health related functional markers.

The Florida manatee’s resilience to anthropogenic

and environmental pressures, coupled with the isolation

from surrounding manatee populations by distance and

open bodies of water, has led to interesting biological

responses on the organismal level. For example, Florida

manatees respond to cold temperatures by amassing in

large numbers at natural and industrial warm water sites.

Manatees in large aggregations are in close proximity to

one another, increasing the potential for disease

transmission through direct contact and coprophagy.

These discrete wintering populations can undergo

fluctuations in numbers resulting in lower genetic

diversity, are vulnerable to potential catastrophic events

and may not have the genetic make-up or resilience to



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number tandem repeats (VNTRs), are sequences of di-,

tri-, tetra-, or larger tandem nucleotides and are an

excellent tool for determining relatedness among

individuals and populations. Each microsatellite locus

can vary in length among individuals. Microsatellites are

neutral markers (not under the influence of selection)

passed down from both parents that provide historical

and contemporary information on relatedness and

population structure. They can also be used to identify

individuals in DNA profiling (sometimes referred to as

“fingerprinting”). This trait has been particularly

beneficial in mark-recapture studies, where genetic

samples acquired through time from individuals is

matched to life-history observations. Recently, this

technique has been applied in the “cradle to grave”

tracking concept used in modeling survivability and

cause of death. It can also provide additional demo-

graphic data, such as age of first reproduction and

paternal component. Additionally, microsatellite

analyses can identify an individuals’ genetic diversity in

relation to health status classification and mortality

events (pers. comm. M. Tringali, FWC). Microsatellites

are helpful in comparing allelic diversity among

populations, determining effective population size, and

calculating the size of an original founder population (1,

22).

Microsatellite studies have been conducted

extensively on the Florida manatee population to

determine spatial structure (23, 62, 78). Highly

polymorphic microsatellite loci are difficult to identify

in manatee tissue samples. Currently, 36 microsatellite

loci have been developed for Florida manatee

genotyping, however only a limited number of alleles

have been identified (23, 36, 62, 77) and all but two are

dinucleotide repeats, increasing scoring difficulty. An

additional 17 primers, designed originally for dugong

populations, were determined to be polymorphic in

Florida manatees as well (36). In the studies conducted

on the Florida manatee the average number of alleles per

locus has been low (Na=2.9,(23); Na=2.8,(62); Na=2.5,

(77); Na=4.2, (78)) when compared to other healthy

mammalian populations. This low allelic diversity in the

Florida manatee and other manatee populations may

decrease the accuracy of population differentiation,

pedigree analyses, and individual assignment to

populations. The identification of more diverse

microsatellite loci may allow for pedigree fingerprinting

11

Allozymes

Previously, allozyme analysis (variation of specific

enzymes) was performed on the Florida manatee to

examine geographic distribution patterns in five areas of

Florida corresponding to carcass recovery locations (49).

Due to the homozygosity observed in this study, the

authors suggested subtle differentiation between each

Florida coast population of manatees, with the likelihood

of some movement among the five regions sampled.

Mitochondrial DNA

Matrilineal haplotypes from sequenced mtDNA

can yield information on phylogenetic and evolutionary

processes. The mitochondrial control region has been

used consistently for studies of manatee populations (24,

48, 82). A 410 bp segment has been most widely used

and no variation in this segment has been detected in

Florida manatees analyzed to date. Cytochrome b (cyt b)

analysis has also been implemented, and illustrated little

variation in the Florida population, but the sample size

was small (12). Additional employment of cyt b studies

would have significant bearing on our understanding of

evolution and lineages between many manatee

populations. Greater diversity in Antillean manatee

samples has illustrated the potential for use of cyt b to

detect phylogeographical units (82). Sequencing other

regions of the mitochondrial genome, such as the

cytochrome c oxidase 1 (CO1) gene, has also been

integrated into the genetic toolbox.

New sequencing technology that makes mapping

of the entire Florida manatee mitogenome possible is

providing additional leads for determining genetic

variation among subpopulations. Additionally, this

information will confer evidence of female gene flow,

philopatry, and lineages to compare and contrast with

results obtained from nuclear DNA studies. The

Antillean manatee mitogenome was sequenced recently

and will have value in comparative research among

sirenian species (2). Complete mapping of genomic

mtDNA in killer whales (Orcinus orca), for example,

has provided information on variations among

populations that have led to discrimination between

three ecotypes within the species (53).

Microsatellites

Microsatellites, also known as simple sequence

repeats (SSRs), short tandem repeats (STRs), or variable



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reproductive fitness (7, 64). The variations in gene

expression among individuals and populations can also

provide information on demographics and adaptive

responses.

Behavioral responses to density-dependent

pressures may affect breeding success, reproduction,

dispersal, and disease susceptibility, which can be

tracked using genetics. Information such as this can help

us gauge the cumulative impacts of climate change (75).

Using advances in health assessment techniques will

allow scientists to develop predictive modeling programs

and study target animal response, which will enable a

more informed approach for management.

The development of manatee specific

genetic assays can provide information on the levels of

gene expression of manatees encountering various

environmental stressors, such as red tide (10), cold stress

(11), and exposure to pollutants (73). Research to detect

gene expression can utilize special microarray chips with

unique sequences. These chips are coated with several

thousand cDNA probes and when linked with a sample

will generate a signal if hybridization occurs. Gene

expression research becomes even more pressing as the

manatee population in Florida responds to a reduction in

available warm water sites with reduced spring flows or

accessibility and the imminent closing of power plants

which provide artificial winter refugia for much of the

Florida manatee population (43, 69). It is uncertain when

artificial sources of warm water currently available for

Florida manatees’ use will finally disappear, but it will

most likely happen within the next couple of decades

(43).

Use of field tests could benefit diagnostic studies

during wild manatee health and risk assessments. For

example, a manatee caught in 2004 in Port of the

Islands, Florida, suffered from a massive internal

infection undetected at the time of capture. Recently,

tools have been designed for diagnosticians to detect

inflammation in manatees using levels of serum amyloid

A (SAA), but assays are expensive and must be

performed at a later time in the lab (29). The manatee

captured in 2004 was released with a radio tag but,

unfortunately, died one month later. Had real-time

information on the acute level of SAA been available in

2004 and during the examination of the manatee in the

field, that manatee could have been taken to a

12

studies that are more precise.

Microsatellites have also been used to address

whether gene flow in Florida manatees is occurring with

other manatee populations (14, 35, 37, 42, 56, 82).

Microsatellite studies of other threatened West Indian

manatee populations are needed urgently to help

determine their relatedness and conservation status.

SNPs

Single nucleotide polymorphisms (SNPs) are

nucleotide variations at a single base which can also aid

in population genetic studies. SNPs are bi-parentally

inherited and can be identified in both intron

(noncoding) and exon (protein-coding) regions. The

presence and uniqueness of polymorphic loci can yield

information on taxonomy and individual identification,

and have proven useful in identifying populations with

low genetic diversity (83). Furthermore, restriction-site

associated DNA (RAD) studies can identify gene

sequences and large numbers of individual SNP sites (4).

Identification of SNPs from genes could as well provide

functional information in addition to detecting

subpopulations for demographic analysis, enabling

managers to implement more effective conservation

measures.

Gene Expression

Functional genomics applies to the parts of the

genome that are actively expressing genes. These

processes result in protein production and relate to the

ability of an individual to respond to biochemical input,

as well as environmental and health challenges.

Examination of gene expression and regulation in

manatees can provide insight into health, reproductive

fitness, and early disease detection. Serial analysis of

gene expression (SAGE) is an approach for analyzing

the observed variation in gene expression among

individuals and populations (81). Understanding

manatee immune genes, susceptibility to disease, wound

healing, bone resorption, dental or other adaptations, and

response to cold or hazardous algal blooms will inform

researchers and managers so they may better monitor

population health status. Also, examination of major

histocompatibility complex (MHC) genes can increase

our understanding of population differences, genetic

diversity, disease defense, mate selection, and



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karyotype has also been employed in evolutionary and

taxonomic studies of sirenians through zoo-Fluorescent

In Situ Hybridization (zoo-FISH) on chromosomes from

other species (41, 42, 60) confirming their affiliation to

the Paenungulata and Afrotheria groups.

Future studies include determining the karyotype

of the West African manatee and the potential for

hybridization between West Indian manatees and

Amazonian manatees at the mouth of the Amazon River

in Brazil (48, 82) will be very important for

understanding sirenian evolution. Knowledge of

interspecific and intraspecific comparisons of

chromosomes between sirenian karyotypes and

ideograms will be beneficial for future studies

determining genome mapping, human (HSA) region

definitions, and banding characteristics.

Implications for Manatee Conservation

Genetic Samples

Current genetic analyses of many manatee

populations in the Trichechidae are incomplete because

of inadequate sample size. Manatee sample collection

has evolved from opportunistic collection of carcasses

and stranded manatees to live animal biopsy. Live

manatee tissue sampling techniques have improved from

the use of cattle ear notchers applied to tail margins of

free-swimming manatees (USGS unpublished data),

dermal skin scrapers (15), biopsy needles attached to

long poles which are employed to sample many

manatees at aggregation sites in winter (16), and health

assessment captures. The collection of fecal samples has

also led to innovative genetic applications in very remote

areas of sirenian distribution where sample collection is

extremely difficult (55, 76). However, use of fecal

material has limitations because the possibility of

contamination, collection of multiple samples from the

same individual, and a lack of DNA in the sample can

cause difficulty in data analysis. There are new

technologies aimed at utilization of fecal samples to

assess hormone and stressor levels (pers. comm. I.

Larkin, UF). Certainly, additional samples influenced by

environmental/endogenous factors obtained from remote

areas throughout the range of manatees and analyzed

with current genetic methods will be useful. Once a

cache of samples is available, a host of options for

13

rehabilitation facility for treatment (30). Additionally,

real-time assessment could be utilized to benefit the

entire local population by informing researchers of links

to potential diseases as well as removing individuals that

may be carriers of disease.

Genomics

Many evolutionarily important species genomes

are currently being sequenced by large-scale initiatives

such as the Genome 10K Project, the Broad Institute and

the Beijing Genomics Institute (31). The complete

Florida manatee whole genome has recently been

shotgun sequenced by the Broad Institute Genome

Assembly and Analysis Group (13). Having this total

genome coverage is extremely beneficial for the species

for collaborative research opportunities and allows

research teams to detect trends in gene expression and

comparisons with other species. Genome-wide

association studies (GWAS), which identify genetic

links to physical or immune traits, could be important in

detecting trends in variability between genetic traits and

disease susceptibility or possibly adaptive potential.

Chromosomes

Individual chromosome homologues for the

Florida manatee (Trichechus manatus latirostris) were

identified using primary chromosome banding

techniques prepared from T- and B-cell peripheral blood

lymphocyte cell cultures established from six individuals

(27). A standard banded karyotype was constructed, for

both sexes, based on the G-banded chromosome pattern

(27). The previously published modal chromosome

number of 48 (47, 84, 85) was confirmed for this

species, whereas, karyotype divergence can be observed

in the Amazonian manatee which supports a model

chromosome number of 56 (3). Digital imaging methods

were subsequently employed and individual homologues

were identified by unique G-banding patterns and

chromosome morphologies. Characterization of

additional cytogenetic features of this species by

supplemental chromosome banding techniques, C-

banding (constitutive heterochromatin), Ag-NOR

staining (nucleolar organizer regions), and DA/DAPI

staining was also performed. Cytogenetic features,

including chromosome morphology and banding

patterns, of T. manatus latirostris were described. The



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the Crystal River manatee population in northwest

Florida when compared to the rest of Florida and cluster

analysis identified two distinct groupings (8). When

samples were compared between the Florida and Belize

manatee populations, very distinct structure was

observed (35), as expected, since the two groups

represent subspecies. Further, the Endangered Species

Act in the U.S. only recognizes the West Indian manatee

throughout its range as one all-inclusive group for legal

protection (79), thereby raising questions as to whether

recovery of a population in one region (e.g., Florida)

implies recovery at the species level of another isolated

population, and subspecies, elsewhere (e.g., Puerto Rico;

37). Thus, one benefit of genetic scrutiny may be in

assisting with the development of separate management

strategies by responsible agencies for each subspecies or

distinct population.

Geographic information currently is used when

selecting release sites for captive born and rehabilitated

manatees, to ensure that manatees from one area of

Florida, or whose parentage is geographically identified,

are not inappropriately introduced into another area.

Presently, Florida manatees typically are not relocated

intentionally from one coast to another, although some

exceptions have occurred in the past (USFWS FMT

meeting notes, USFWS Captive Manatee Database,

USGS files). Generally, captive manatees are released to

populations that have similar characteristics, with the

expectation of preserving beneficial local adaptations. In

some cases reduced genetic diversity could have positive

impacts on the resilience of local populations to face

perturbations. For example, manatees may be better

suited to certain environmental or habitat characteristics,

such as the strong influence of red tide on west coast

manatees which may have resulted in locally-adapted

gene complexes. Relocating manatees from other areas

can result in outbreeding depression where the offspring

are poorly suited to the habitat and the ability to produce

important gene complexes. Alternatively, breeding with

relocated individuals can result in genetic swamping,

where the new genetic signature quickly become fixed in

the population, replacing the locally-adapted alleles.

Information on manatee effective population size

in Florida could be useful for integration into the Core

Biological Model (CBM) for determining population

viability (68). The CBM is a useful tool for assessing

14

genetic analyses exists.

Implementing Molecular Tools in Conservation

Genetics

Currently, no standards or guidelines are in place

to assist researchers and managers with the appropriate

selection of genetic tools to study the status of wild

populations (19). As each population of wildlife is

unique and warrants different considerations,

standardization among species is problematic. The

information gained through additional genetic analyses,

however, would greatly enhance the possibilities for

preservation of the populations of imperiled populations.

The discreteness (amount) and significance (type)

of genetic analysis must be appropriate for the questions

to be addressed (19). Microsatellites have been useful

for delimiting population differences in many species

(17, 26, 39, 61, 65, 70). Species that have been isolated

for long periods of time generally require smaller data

sets or fewer markers for analysis, whereas recent

separation of populations (such as that which has

happened in the Florida manatee subspecies) usually

requires more samples and the application of multiple

genetic techniques (19). Generally, multiple genetic

markers versus a single type of marker are favored, but

care should be exercised when selecting the appropriate

set of markers. Studies have demonstrated that a more

robust assessment is obtained when more markers are

employed (46). Neutral markers, versus genes that code

for adaptive variation under selection in the

environment, change more quickly, making them good

indicators of population uniqueness (34, 50, 51). Care

must be taken to ensure that the genetic data are used in

conjunction with existing ecologic, geographic,

morphologic, and life history data sets (28).

Technologies and tools for molecular analyses

continue to evolve and many currently are applied to

understand the Florida manatee population. Allozymes,

mtDNA, and microsatellites demonstrated little to no

genetic differences among the four existing Florida

manatee MUs (12, 25, 49, 63, 82), however recent

analyses using new spatial statistical techniques are

suggesting differences among these four MUs, but more

specifically the populations inhabiting the east and west

coasts of Florida (pers. comm. M. Tringali, FWC). This

difference between the coasts also was supported when



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identification of individuals independent of typical

scarring features presently used and essential to confirm

identities and lineages. Genetic tools would be useful for

studies of other trichechid and dugongid populations as

well. The benefits from engaging conservation genetics

tools could strengthen important conservation decision

processes. Scientific information is the primary

component to facilitate effective conservation practices.

The future direction of genetic research will utilize

sequencing modalities, implement large volumes of data,

and will design tools to gauge the health and fitness of

individuals within all populations of sirenians. New

initiatives to embrace genetic health and risk assessment

on wild sirenian populations may give researchers a

more effective way of assessing populations and gauging

recovery status (9). Such new information will be useful

to managers for implementation in manatee management

and protection strategies world-wide.

Disclaimer

Any use of trade, product, or firm names is for

descriptive purposes only and does not imply

endorsement by the U.S. Government.

References

1. Allendorf FW, Luikart G. Conservation and

Genetics of Populations. Blackwell Publishing,

Malden, Massachusetts. 642 pp. 2007.

2. Arnason U, Adegoke JA, Gullberg A, Harley EH,

Janke A, Kullberg M. Mitogenomic relationships

of placental mammals and molecular estimates of

their divergences. Gene 421:37-51. 2008.

3. Assis, MFL, Best RC, Barros RMS,

Yonenaga-Yassuda Y. Cytogenetic study of

Trichechus inunguis (Amazonian manatee).

Revista Brasileira de Genitica 11:41-50. 1988.

4. Baird NA, Etter PD, Atwood TS, Currey MC,

Shiver AL, Lewis ZA, et al. Rapid SNP discovery

and genetic mapping using sequenced RAD

markers. Public Library of Science One

3(10):e3376. 2008.

5. Beaumont MA. Estimation of population growth

or decline in genetically monitored populations.

Genetics 164:139-160. 2003.

15

manatee population status based upon current

understanding of annual variability in survival and

reproductive rates, demographic stochasticity (including

changes in effective population size), effects of changes

in warm-water availability, and catastrophes (69). As we

are aware, a reduction in effective population size is a

loss of genetic diversity (1, 22), as has been observed in

the Florida manatee; this loss of diversity in populations

may result in loss of the ability to adapt to environmental

changes (20, 21). Therefore, greater effort could usefully

be given to improve the effective population size in the

manatee. As the human population continues to grow,

impacts on the environment and available resources

becomes even greater, creating a need for rigorous

scientific information on affected wildlife species (67).

Recent genetic findings have illustrated that there

is low allelic diversity in the manatee population in

Florida (62) which is likely the result of a founder event

that occurred during the Pleistocene epoch. Additionally,

Florida manatees have a low effective population (Ne)

(78). This raises concerns for the well-being of the

manatee population as a whole in Florida, as many

policies regarding their protection are based primarily on

estimated total population size. Additional studies are

needed to examine population fitness and the impacts of

inbreeding in the Florida manatee. Studies have shown

that once a population drops below inbreeding

thresholds, detrimental effects can occur during

stochastic change (22). Continued examination of

genetic markers would be very informative for

determining the resiliency of manatee populations to

perturbations leading to dramatic population

fluctuations. These population increases and declines

have occurred in the resident manatee population as a

whole in Florida during periods of existence with

humans (59). Genetic markers can also be utilized to

determine evolutionary lineages with predictions of

founder population size and subsequent coalescent time

calculation dating back to the event (5).

Presently, manatees with distinct scarring are

photographed (6) and that information is used in

mark-recapture modeling efforts throughout Florida (44,

45). Integration of genetic-identification of individuals

with conventional photo-identification technology will

provide additional sources for capture-recapture

analyses. These additional genetic tools will facilitate the



JMATE

6. Beck CA, Reid JP. An automated photo-

identification catalog for studies of the life

history of the Florida manatee. In: Population

Biology of the Florida Manatee. (eds. O’Shea TJ,

Ackerman BB, Percival HF), pp. 120-134.

National Biological Service Information and

Technology Report 1. Washington, D. C. 1995.

7. Bernatchez L, Landry C. MHC studies in non-

model vertebrates: what have we learned about

natural selection in 15 years? Journal of

Evolutionary Biology 16:363-377. 2003.

8. Bonde RK. Population genetics and conservation

of the Florida manatee: past, present, and future.

PhD Dissertation, University of Florida,

Gainesville, Florida. 144 pp. 2009.

9. Bonde, RK, Aguirre AA, Powell J. Manatees as

sentinels of marine ecosystem health: are they the

2000-pound canaries? EcoHealth 1:255-262. 2004.

10. Bossart GD, Baden DG, Ewing RY, Roberts B,

Wright SD. Brevetoxicosis in manatees

(Trichechus manatus latirostris) from the 1996

epizootic: gross, histologic and

immunohistochemical features. Toxicologic

Pathology 26:276-282. 1998.

11. Bossart GD, Meisner RA, Rommel SA, Ghim S,

Jenson AB. Pathological features of the Florida

manatee cold stress syndrome. Aquatic Mammals

29:9-17. 2002.

12. Bradley JL, Wright SD, McGuire PM. The

Florida manatee: cytochrome-b DNA sequence.

Marine Mammal Science 9:197-202. 1993.

13. Broad Institute Genome Assembly and Analyses

Group. Accessed 07 August 2012. Available from

http://www.broadinstitute.org/software/allpaths-lg/

blog/?p=440. 2012.

14. Cantanhede AM, da Silva VMF, Farias IP, Hrbek

T, Lazzarinis SM, Alves-Gomez J.

Phylogeography and population genetics of the

endangered Amazonian manatee, Trichechus

inunguis Natterer, 1883 (Mammalia, Sirenia).

Molecular Ecology 14:401-413. 2005.

15. Carney SL, Bolton EE, Barton SL, Scolardi KM,

Englund CC, Tringali MD, et al. A minimally

invasive method of field sampling for genetic

analyses of the Florida manatee (Trichechus

manatus latirostris). Marine Mammal Science

23:967-975. 2007.

16. Davis MC, Seyoum S, Carney SL, Rodriguez-

Lopez MA, Bolin EE, Sullivan JG, et al. This

won't hurt a bit: new molecular tools for

population assessment of the Florida manatee. Ab-

stract. Eighteenth Biennial Conference on the Bi-

ology of Marine Mammals, 12-16 October 2009,

Quebec, Canada. 2009.

17. Delgado JV, Martínez AM, Acosta A, Álvarez LA,

Armstrong E, Camacho E, et al. Genetic

characterization of Latin-American Creole cattle

using microsatellite markers. Animal Genetics

43:2-10. 2012.

18. Dizon AE, Lockyear C, Perrin W, Demaster DP,

Sisson J. Rethinking the stock concept: a

phylogeographic approach. Conservation Biology

6:24-36. 1992.

19. Fallon SM. Genetic data and the listing of

species under the U.S. Endangered Species Act.

Conservation Biology 21:1186-1195. 2007.

20. Franklin IR, Frankham R. How large must

populations be to retain evolutionary potential?

Animal Conservation 1:69-73. 1998.

21. Frankham R. Inbreeding and extinction - a

threshold effect. Conservation Biology 9:792-799.

1995.

22. Frankham R, Ballou JD, Briscoe DA.

Introduction to Conservation Genetics.

Cambridge University Press, Cambridge, UK. 640

pp. 2002.

23. Garcia-Rodriguez AI. Genetic studies of the West

Indian manatee (Trichechus manatus). PhD

dissertation, University of Florida, Gainsville,

Florida. 115 pp. 2000.

24. Garcia-Rodriguez AI, Bowen BW, Domning D,

Mignucci-Giannoni AA, Marmontel M, Montoya-

Ospina RA, et al. Phylogeography of the West In-

dian manatee (Trichechus manatus): how many

populations and how many taxa? Molecular Ecol-

ogy 7:1137-1149. 1998.

25. Garcia-Rodriguez AI, Moraga-Amador D,

Farmerie W, McGuire P, King TL. Isolation and

characterization of microsatellite DNA markers in

the Florida manatee (Trichechus manatus

latirostris) and their application in Sirenian

species. Molecular Ecology 9:2161-2163. 2000.

16



JMATE

26. Goldstein DB, Pollock PD. Launching

microsatellites: a review of mutation processes and

methods of phylogenetic inference. Journal of

Heredity 88:335-342. 1997.

27. Gray BA, Zori RT, McGuire PM, Bonde RK. A

first generation cytogenetic ideogram for the

Florida manatee (Trichechus manatus latirostris)

based on multiple chromosome banding

techniques. Hereditas 137:215-223. 2002.

28. Haig SM, Beever EA, Chambers SM, Draheim

DM, Dugger BD, Dunham S, et al. Taxonomic

considerations in listing subspecies under the U.S.

Endangered Species Act. Conservation Biology

20:1584-1594. 2006.

29. Harr KE, Allison K, Bonde RK, Murphy D,

Harvey JW. Comparison of blood

aminotransferase methods for assessment of

myopathy and hepatopathy in Florida manatees

(Trichechus manatus latirostris). Journal of Zoo

and Wildlife Medicine 39:180-187. 2006.

30. Harr KE, Rember R, Ginn PE, Lightsey J, Keller

M, Reid J, et al. Chronic boat strike trauma and

polycystic kidneys in a wild Florida manatee

(Trichechus manatus latirostris). Journal of

Wildlife Diseases 47:1026-1031. 2011.

31. Haussler D, O’Brien SJ, Ryder OA. Genome 10K:

a proposal to obtain whole-genome sequence for

10,000 vertebrate species. Journal of Heredity

100:659-674. 2009.

32. Herbert PDN, Stoeckle MY, Zemlak TS, Francis

CM. Identification of birds through DNA

barcodes. Public Library of Science Biology 2

(10):e312. 2004.

33. Hoelzel AR, Halley J, O'Brien SJ, Campagna C,

Arnborm T, Le Boeuf B, et al. Elephant seal

genetic variation and the use of simulation-models

to investigate historical population bottlenecks.

Journal of Heredity 84:443-449. 1993.

34. Hoekstra HE, Drumm KE, Nachman NW.

Ecological genetics of adaptive color

polymorphism in pocket mice: geographic

variation in selected and neutral genes. Evolution

58:1329-1341. 2004.

35. Hunter ME, Auil-Gomez NE, Tucker KP, Bonde

RK, Powell J, McGuire PM. Little genetic

variation in the regionally important Belize

manatee and evidence of low intra-populational

and sub-species dispersal. Animal Conservation

13:592-602. 2010.

36. Hunter M, Broderick D, Ovenden J, Tucker KP,

Bonde RK, McGuire PM, et al. Characterization

of highly informative cross-species microsatellite

panels for the Australian dugong (Dugong

dugon) and Florida manatee (Trichechus

manatus latirostris) including five novel primers.

Molecular Ecology Resources 10:368-377. 2010.

37. Hunter ME, Mignucci-Giannoni AA, King TL,

Tucker KP, Bonde RK, McGuire PM.

Comprehensive genetic investigation recognizes

evolutionary divergence in the Florida

(Trichechus manatus latirostris) and Puerto Rico

(T. m. manatus) manatee populations and subtle

substructure in Puerto Rico. Conservation

Genetics in press. 2012.

38. IUCN. 2009.1 IUCN Red List of Threatened

Species. Accessed 01 April 2010. Available from

http://www.iucnredlist.org/. 2009.

39. Jarne P, Lagoda PJL. Microsatellites, from

molecules to populations and back. Trends in

Ecology & Evolution 11:424-429. 1996.

40. Johnson WE, Onorato DP, Roelke ME, Land DE,

Cunningham M, Belden RC, et al. Genetic

restoration of the Florida panther. Science

329:1641-1645. 2010.

41. Kellogg ME. Sirenian conservation genetics and

Florida manatee (Trichechus manatus latirostris)

cytogenetics. PhD Dissertation, University of

Florida, Gainesville, Florida. 159 pp. 2008.

42 Kellogg ME, Burkett S, Dennis TR, Stone G,

Gray BA, McGuire PM, et al. Chromosome paint-

ing in the manatee supports Afrotheria and Pae-

nungulata. BMC Evolutionary Biology

7(6):1-7. 2007.

43. Laist DW, Reynolds III, JE. Influence of power

plants and other warm-water refuges on Florida

manatees. Marine Mammal Science 21:739-764.

2005.

44. Langtimm CA, Beck CA. Lower survival

probabilities for adult Florida manatees in years

with intense coastal storms. Ecological

Applications 13:257-268. 2003.

17



JMATE

45. Langtimm CA, Beck CA, Edwards HH,

Fick-Child KJ, Ackerman BB, Barton SL, et al.

Survival estimates for Florida manatees from the

photo-identification of individuals. Marine

Mammal Science 20:438-463. 2004.

46. Latta RG. Integrating patterns across multiple

genetic markers to infer spatial processes.

Landscape Ecology 21:809-820. 2006.

47. Loughman W, Frye F, Herald E. The

chromosomes of a male manatee. International

Zoo Yearbook 10:151-152. 1970.

48. Luna FO, Bonde RK, Attademo FN, Saunders JW,

Meigs-Friend G, Passavante JZO, et al.

Phylogeographic implications for release of

critically endangered manatee calves rescued in

Northeast Brazil. Aquatic Conservation: Marine

and Freshwater Ecosystems 22(5):2012. 4 JUL

2012, DOI: 10.1002/aqc.2260

49. McClenaghan LR, O'Shea TJ. Genetic variability

in the Florida manatee (Trichechus

manatus). Journal of Mammalogy 69:481-488.

1988.

50. McKay JK, Latta RG. Adaptive population

divergence: markers, QTL and traits. Trends in

Ecology & Evolution 17:285-291. 2002.

51. Merila J, Crnokrak P. Comparison of genetic

differentiation at marker loci and quantitative

traits. Journal of Evolutionary Biology

14:892–903. 2001.

52. Metzker, ML. Sequencing technologies - the next

generation. Nature Reviews/Genetics 11:31-46.

2010.

53 Morin PA, Allen E, Pitman R, Li L, Bouffard P,

Foote A, et al. Population mitogenomics of killer

whales: 2.5 million bp of DNA sequence yields

global patterns of killer whale ecotype evolution.

Abstract. Eighteenth Biennial Conference on the

Biology of Marine Mammals, 12-16 October 2009,

Quebec, Canada. 2009.

54. Munoz-Vinas S. Contemporary Theory of

Conservation 2nd edition. Butterworth-Heinemann

Publishers, Oxford, United Kingdom. 256 pp.

2004.

55. Muschett G, Bonacic C, Vianna JA. A

noninvasive sampling method for genetic

analysis of the West Indian manatee (Trichechus

manatus). Marine Mammal Science 25:955-963.

2009.

56. Nourisson C, Morales-Vela B, Padilla-Saldivar J,

Tucker KP, Clark AM, Olivera LD, et al.

Evidence of two genetic clusters of manatees with

low genetic diversity in Mexico and

implications for their conservation. Genetica

139:833-842. 2011.

57. O’Brien SJ. Tears of the Cheetah. Saint Martin’s

Press, New York, New York. 287 pp. 2003.

58. O’Brien SJ, Roelke M, Marker L, Newman A,

Winkler CA, Meltzer DG, et al. Genetic basis for

species vulnerability in the cheetah. Science

227:1428-1434. 1985.

59. O'Shea TJ. The past, present, and future of

manatees in the southeastern United States:

realities, misunderstandings, and enigmas. In:

Proceedings of the Third Southeastern Nongame

and Endangered Wildlife Symposium. Georgia

Department of Natural Resources, Game and Fish

Division, Social Circle, Georgia. 253 pp. 1988.

60. Pardini AT, O'Brien PCM, Fu B, Bonde RK, Elder

FFB, Ferguson-Smith MA, et al. Chromosome

painting among Proboscidea, Hyracoidea and

Sirenia: support for Paenungulata (Afrotheria,

Mammalia) but not Tethytheria. Proceedings of

the Royal Society B: Biological Sciences

274:1333-1340. 2007.

61. Patterson N, Price AL, Reich D. Population

structure and eigenanalysis. Public Library of

Science Genetics 2(12):e190. 2006.

62. Pause, K.C. Conservation genetics of the Florida

manatee, Trichechus manatus latirostris. PhD

Dissertation, University of Florida, Gainesville,

Florida. 177 pp. 2007.

63. Pause KC, Nourisson C, Clark A, Kellogg ME,

Bonde RK, McGuire PM. Polymorphic

microsatellite DNA markers for the Florida

manatee (Trichechus manatus latirostris).

Molecular Ecology Notes 7:1073-1076. 2007.

64. Piertney SB, Oliver MK. The evolutionary

ecology of the major histocompatibility complex.

Heredity 96:7-21. 2006.

65. Quellar DC, Strassmann JE, Hughes CR.

Microsatellites and kinship. Trends in Ecology &

Evolution 8:285-288. 1993.

18



JMATE

66. Quintana-Rizzo E, Reynolds III JE. Regional

management plan for the West Indian manatee

(Trichechus manatus). Caribbean Environment

Programme, United Nations Environment

Programme, CEP Technical Report #48. 2010.

67. Reep RL, Bonde RK. The Florida Manatee:

Biology and Conservation. University Press of

Florida, Gainesville, Florida. 224 pp. 2006.

68. Runge MC, Sanders-Reed CA, Fonnesbeck CJ. A

core stochastic projection model for Florida

manatees (Trichechus manatus latirostris). U.S.

Geological Survey Open-File Report 2007-1082.

41 pp. 2007.

69. Runge MC, Sanders-Reed CA, Langtimm CA,

Fonnesbeck CJ. A quantitative threats analysis for

the Florida manatee (Trichechus manatus

latirostris). U.S. Geological Survey Open-File

Report 2007-1086. 34 pp. 2007.

70. Seki Y, Yokohama M, Ishikawa D, Ikehara N,

Wada K, Nomura K, et al. Development and

characterization of 260 microsatellite loci in the

domestic goat, Capra hircus. Animal Genetics

43:365-366. 2012.

71. Slade RW, Moritz C, Hoelzel AR, Burton HR.

Molecular population genetics of the southern

elephant seal Mirounga leonina. Genetics

149:1945-1957. 1998.

72. Stejneger L. How the great northern sea cow

(Rytina) became exterminated. American

Naturalist 21:1047-1056. 1887.

73. Stockwell BR. Chemical genetic screening

approaches to neurobiology. Neuron 36:559-562.

2003.

74. Stoeckle M. Taxonomy, DNA and the bar code of

life. BioScience 53:2-3. 2003.

75. Thomas CD, Cameron A, Green RE, Bakkenes M,

Beaumont LJ, Collingham YC, et al.

Extinction risk from climate change. Nature

427:145-148. 2004.

76. Tikel D, Blair D, Marsh HD. Marine mammal fae-

ces as a source of DNA. Molecular Ecology

5:456-457. 1994.

77. Tringali MD, Seyoum S, Carney SL, Davis MC,

Rodriguez-Lopez MA, Reynolds III JE, et al.

Eighteen new polymorphic microsatellite

markers for the endangered Florida manatee,

Trichechus manatus latirostris. Molecular

Ecology Resources 8:328-331. 2008.

78. Tucker KP, Hunter ME, Bonde RK, Austin JD,

Clark AM, Beck CA, et al. Genetically defined

demographic parameters to improve threat

analyses and management decisions in the

endangered Florida manatee. Journal of

Mammalogy, 93(6): in press. 2012.

79. USFWS. West Indian Manatee (Trichechus

manatus) 5-Year Review: Summary and

Evaluation. USFWS, Southeastern Region,

Atlanta, Georgia. 79 pp. 2007.

80. USFWS. Endangered Species Program. Accessed

07 Aug 2012. Available from http://

www.fws.gov/endangered/. 2012.

81. Velculescu VE, Zhang L, Vogelstein B, Kinzler

KW. Serial analysis of gene expression. Science

270:484-487. 1995.

82. Vianna JA, Bonde RK, Caballero S, Giraldo JP,

Lima RP, Clark A, et al. Phylogeography,

phylogeny and hybridization in trichechid

sirenians: implications for manatee

conservation. Molecular Ecology 15:433-447.

2006.

83. Vignal A, Milan D, SanCristobal M, Eggan A. A

review of SNP and other types of molecular

markers and their use in animal genetics.

Genetics Selection Evolution 34:275-305. 2002.

84. White JR, Harkness DR, Isaacks RE, Duffield

DA. Some studies on blood of the Florida

manatee, Trichechus manatus latirostris.

Comparative Biochemistry & Physiology – Part

A 55:413-417. 1976.

85. White JR, Harkness DR, Isaacks RE, Duffield

DA. Trichechus manatus latirostris (manatee).

Order: Sirenia. Family: Trichechidae. In: An

Atlas of Mammalian Chromosomes (eds T. C. Hsu

and K. Benirschke). Springer-Verlag, Folio. 496

pp. 1977.

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